The present invention relates to a 1-aryl-1,8-naphthylidin-4-one derivative as a selective type IV phosphodiesterase (i.e., xe2x80x9cPDExe2x80x9d) inhibitor and a salt, and solvate thereof and a pharmaceutical composition and a type IV phosphodiesterase inhibitor containing the same as an effective component as well as an agent of preventing or treating for cytokine related diseases.
The intracellular second messenger cAMP or cGMP is broken down and deactivated by phosphodiesterase (PDE), which is classified into at least types I to VII. PDE is widely distributed in the tissue and organs of the body. Among these, type IV phosphodiesterase selectively breaks down cAMP and is found in the central tissue and in the heart, lungs, kidneys, and other organs and in the various hemocyte components etc. Further, it is known to be involved in the derivation of IL-1 and IL-6, TNF-xcex1, and other various cytokines.
Catechol type derivatives such as rolipram, known to be a selective inhibitor of this enzyme, quinazoline type derivatives such as nitraquazone, xanthine type derivatives such as theophylline and denbufylline, etc. are being used or developed as antidepressants, antiasthmatics, antiinflamatorics, etc. No drug has however yet been developed which solves the problems such as the selectivity with other isoenzymes and various side effects. There is no satisfactory medicine which has this enzyme inhibiting action as the main mechanism for achieving the medicinal effect.
On the other hand, as a compound having a PDE IV inhibiting action and a naphthylidinone skeleton, for example, as a compound having a carbonyl group at the 2-position in the 1,8-naphthylidine skeleton, there are known those described in JP-A-55-164682, WO-A-94-12499, WO-A-96-06843, etc.
Further, as a compound having a PDE IV inhibiting action and carbonyl group at the 4-position in a 1,8-naphthylidine skeleton, WO-A-97-04775 describes one where the 1-position substituent group is an ethyl group. Further, as the method of synthesis described in this publication, the method shown in the following formula was used, based on the method of Kaminsky et al. (J. Med. Chem. 1968, 11, 160). However, the 1-position substituent group disclosed in this method is only an alkyl group. 
In the above reaction process, it is only possible to use a substitution reaction using a highly reactive alkyl halide (Axe2x80x94CH2xe2x80x94Y), and therefore, the substituent groups which can be introduced to the 1-position are limited.
The objects of the present invention are to provide a compound or a salt or solvate thereof, useful as a medicine for the prevention or treatment of bronchial asthma, chronic bronchitis, and other respiratory diseases, diseases relating to abnormality of nervous system such as impaired learning, memory, and recognition relating to Alzheimer""s disease, Parkinson""s disease, and the like, diseases relating to mental abnormality such as maniac depression and schizophrenia, atbpic dermitis, conjunctivitis, acquired immunity disorder syndrome and other inflammatory diseases, osteoarthritis, rheumatoid arthritis, and other general or local joint diseases, rheumatoid arthritis, sepsis, Crohn disease and other diseases which are related to various cytokines such as tumor necrosis factor (TNF-xcex1), and the like by selectively inhibiting the type IV phosphodiesterase and further inhibiting the production of TNF-xcex1.
In accordance with the present invention, there is provided a 1-aryl-1,8-naphthylidin-4-one derivative having the formula (I): 
wherein R1 indicates a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group,
R2, R3, and R4 independently indicate a hydrogen atom, a substituted or unsubstituted lower alkyl group, or a halogen atom,
X indicates the group NR5R6 or group OR7, wherein R5 and R6 independently indicate a hydrogen atom, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group, and R7 indicates a hydrogen atom, a substituted or unsubstituted lower alkyl group, or a substituted or unsubstituted cycloalkyl group or a salt or solvate thereof.
In accordance with the present invention, there is also provided a 1-aryl-1,8-naphthylidin-4-one derivative having the formula (Ixe2x80x2): 
wherein R1 indicates a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group,
R2xe2x80x2, R3xe2x80x2, and R4xe2x80x2 independently indicate a hydrogen atom, or a substituted or unsubstituted lower alkyl group,
Xxe2x80x2 indicates the group NR5R6,
R5 and R6 independently indicate a hydrogen atom, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group
or a salt or solvate thereof.
In accordance with the present invention, there is also provided a pharmaceutical composition and a type IV phosphodiesterase inhibitor as well as an agent of preventing or treating for cytokine related diseases, containing a 1-aryl-1,8-naphthylidin-4-one derivative as set forth in the above formula (I) or (Ixe2x80x2) or its pharmaceutically acceptable salt or solvate as the effective ingredient.
Further, according to the present invention, it is possible to provide a synthesis intermediate useful for the production of a 1-aryl-1,8-naphthylidin-4-one derivative as set forth in the above general formula (I) or (Ixe2x80x2).
The inventors engaged in intensive research to develop a compound having a superior type IV phosphodiesterase inhibiting action and a process for producing the same and, as a result, found that a compound having the formula (I) or (Ixe2x80x2) with a carbonyl group at the 4-position in the 1,8-naphthylidine skeleton and an aryl group or heteroaryl group as the 1-position substituent group has a superior type IV phosphodiesterase inhibiting action, whereby the present invention was completed.
The preferable examples of the aryl group of the substituted or unsubstituted aryl group indicated by R in the formula (I) and (Ixe2x80x2) according to the present invention are C6 to C14 aryl group, for example, a phenyl group, naphthyl group, indenyl group, anthryl group, etc. More preferable example is a phenyl group. Preferable examples of the substituents for the aryl group are a hydroxyl group, a lower alkyl group, a halogen atom such as a fluorine, chlorine, bromine or iodine atom, an oxygen atom, a sulfur atom, an alkoxy group, a cyano group, a nitro group, an amino group, an alkylamino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc.
In the specification, xe2x80x9clowerxe2x80x9d, unless otherwise alluded to, means 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms.
Preferable examples of the heteroaryl group of the substituted or unsubstituted heteroaryl group indicated by R1 in the formula (I) or (Ixe2x80x2) of the present invention are a monocyclic or polycyclic heteroaryl group having a 5- to 7-member ring including 2 to 8 carbon atoms and 1 to 4 hetero atoms of an oxygen atom, a nitrogen atom, or a sulfur atom, for example, a pyrrole group, a furyl group a thienyl group, an imidazolyl group, a thiazolyl group, a pyridyl group, a pyrazinyl group, an indolyl group, a quinolyl group, an isoquinolyl group, etc. may be mentioned. The more preferable example is a pyridyl group. Examples of the preferable substituent groups of the heteroaryl group are ahydroxyl group, a lower alkyl group, a halogen atom such as a fluorine, chlorine, bromine or iodine atom, an oxygen atom, a sulfur atom, an alkoxy group, a cyano group, a nitro group, an amino group, are alkylamino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc.
The preferable examples of the lower alkyl group indicated by R2, R3, or R4 in the formula (I) or R2xe2x80x2, R3xe2x80x2 or R4xe2x80x2 in the formula (Ixe2x80x2) are C1-C6 linear or branched alkyl group such as a methyl group, ethyl group, n-propyl group, isopropyl group, isobutyl group, t-butyl group, etc. The preferable examples of the halogen atom indicated by R2, R3, or R4, in the formula (I), fluorine, chlorine, bromine, and iodine. The preferable examples of a combination of R2, R3, and R4, or a combination of R2xe2x80x2, R3xe2x80x2 and R4xe2x80x2, are all hydrogen atoms.
The preferable examples of the alkyl group of the substituted or unsubstituted lower alkyl group indicated by R5 or R6 in formula (I) or (Ixe2x80x2) are C1 to C6 linear or branched alkyl group such as a methyl group, ethyl group, n-propyl group, isopropyl group, isobutyl group, t-butyl group, etc. The preferable examples of the substituent group of the lower alkyl group are preferably C3 to C6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group, C6 to C14 aryl group such as a phenyl group, lower alkenyl group such as a vinyl group, etc., a halogen atom such as a fluorine, chlorine, bromine or iodine atom, a hydroxyl group, an alkoxy group, a cyano group, a nitro group, an amino group, an alkylamino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc.
The preferable examples of the cycloalkyl group of the substituted or unsubstituted cycloalkyl group indicated by R5 or R6 in formula (I) or (Ixe2x80x2) are C3-C6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. The preferable examples of the substituent group for the cycloalkyl group are a C6 to C14 aryl group such as a phenyl group, lower alkenyl group such as a vinyl group, etc. a hydroxyl group, a halogen atom such as a fluorine, chlorine, bromine or iodine atom, an alkoxy group, a cyano group, a nitro group, an amino group, an alkylamino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc.
The preferable examples of the aryl group of the substituted or unsubstituted aryl group indicated by R5 or R6 in formula (I) or (Ixe2x80x2) are a C6 to C14 aryl group such as a phenyl group, a naphthyl group, an indenyl group, an anthryl group, etc. The more preferable example is a phenyl group. Further, preferable examples of the substituent group for the aryl group are C1-C6 linear or branched alkyl group such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, an isobutyl group and a t-butyl group, a C6-C14 aryl group such as a phenyl group and a naphthyl group, a halogen atom such as fluorine, chlorine, bromine or iodine atom, a hydroxy group, an alkoxy group, a cyano group, a nitro group, an amino group, an alkylamino group, an amido group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc.
Further, preferable examples of the heteroaryl group of the substituted or unsubstituted heteroaryl group indicated by R5 or R6 in formula (I) or (Ixe2x80x2) are a monocyclic or polycyclic heteroaryl group having a 5- to 7-member ring including 1 to 4 hetero atoms including an oxygen atom, a nitrogen atom, or a sulfur atom such as a pyrrole group, a furyl group, a thienyl group, an imidazolyl group, a thiazolyl group, a pyridyl group, a pyrazinyl group, an indolyl group, a quinolyl group, an isoquinolyl group, a benzimidazolyl group, a benzthiazolyl group, etc. More-preferable examples are a 4-pyridyl group, a 3-pyridyl group, a 2-pyridyl group, a thiazolyl group, etc. The examples of preferable substituent groups for the heteroaryl group are a C1-C6 linear of branched alkyl group such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, an isobutyl group, a t-butyl group, a C6-C14 aryl group such as a phenyl group, a naphthyl group, a halogen atom such as fluorine, chlorine, bromine or iodine atom, a hydroxy group, an alkoxy group, a cyano group, a nitro group, an amino group, an alkylamino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc. The preferable examples of the substituted or unsubstituted heteroaryl group indicated by R5 or R6 are a 4-pyridyl group, a 3-pyridyl group, a 3,5-dichloropyridin-4-yl group, etc.
The preferable examples of the lower alkyl group of the substituted or unsubstituted lower alkyl group indicated by R7 in formula (I) are a C1-C6 linear or branched alkyl group such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, an isobutyl group,a t-butyl group, etc. The preferable examples of the substituent group of the lower alkyl group are a C6-C14 aryl group such as a phenyl group, a halogen atom such as fluorine, chlorine, bromine or iodine atom, a lower alkenyl group such as a vinyl group, etc. a cyano group, a nitro group, an amino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group.
The preferable examples of the cycloalkyl group for the substituted or unsubstituted cycloalkyl group indicated by R7 in formula (I) are a C3-C6 cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. The preferable examples of the substituent group for the cycloalkyl group are a C6-C14 aryl group such as a phenyl group, etc. a lower alkenyl group such as a vinyl group, etc. a halogen atom such as a fluorine, chlorine, bromine or iodine atom, a hydroxy group, an alkoxy group, a cyano group, a nitro group, an amino group, an alkylamino group, an amide group, an acyl group, an acyloxy group, a carboxyl group, a lower alkoxycarbonyl group, an aralkyloxycarbonyl group, a carbamoyl group, a thiol group, an alkylthio group, a sulfonyl group, etc.
The specific examples of the 1-aryl-1,8-naphthylidin-4-one derivative described in the above formula (I) or (Ixe2x80x2) of the present invention are as follows.
(a) a 1-aryl-1,8-naphthylidin-4-one derivative where all of R2, R3, and R4 or R2xe2x80x2, R3xe2x80x2 and R4xe2x80x2 are hydrogen atoms
(b) a 1-aryl-1,8-naphthylidin-4-one derivative where R1 is a phenyl group
(c) a 1-aryl-1,8-naphthylidin-4-one derivative where R1 is a tolyl group
(d) a 1-aryl-1,8-naphthylidin-4-one derivative where R1 is an anisil group
(e) a 1-aryl-1,8-naphthylidin-4-one derivative where R1 is a phenyl group substituted with a halogen atom
(f) a 1-aryl-1,8-naphthylidin-4-one derivative where R1 is a pyridyl group
(g) a 1-aryl-1,8-naphthylidin-4-one derivative where R1 is a thiazolyl group
(h) a 1-aryl-1,8-naphthylidin-4-one derivative where one of R5 or R6 is a hydrogen atom
(i) a 1-aryl-1,8-naphthylidin-4-one derivative where one of R5 or RF is 4-pyridyl group and the other is a hydrogen atom
(j) a 1-aryl-1,8-naphthylidin-4-one derivative where one of R5 or R6 is 3-pyridyl group and the other is a hydrogen atom
(k) a 1-aryl-1,8-naphthylidin-4-one derivative where one of R5 or R6 is a 2-pyridyl group and the other is a hydrogen atom
(l) a 1-aryl-1,8-naphthylidin-4-one derivative where one of R5 or R6 is a 2,6-dichlorophenyl group and the other is a hydrogen atom
(m) a 1-aryl-1,8-naphthylidin-4-one derivative where one of R5 or R6 is a 3,5-dichloropyridin-4-yl group and the other is a hydrogen atom
(n) a 1-aryl-1,8-naphthylidin-4-one derivative where X is a group NR5R6 
The compound of the present invention has one or more asymmetric carbon atoms. Based on this, there are (R)-isomers, (S)-isomers, and other optical isomers, racemics, diastereomers, etc. Further, depending on the type of the substituent group, there are double bonds, and therefore, there are also (Z)-isomers, (E)-isomers, and the other geometrical isomers. The present invention includes these isomers separated from each other or in mixtures.
The compounds of the present invention include those capable of forming salts with acids. As the salts, acid addition salts with a mineral acid such as hydrochloric acid, a hydrobromic acid, a hydroiodic acid, a sulfuric acid, a nitric acid, a phosphoric acid, and with an organic acid such as a formic acid, an acetic acid, a propionic acid, an oxalic acid, a malonic acid, a succinic acid, a fumaric acid, a maleic acid, a lactic acid, a malic acid, a citric acid, a tartaric acid, a picric acid, a methanesulfonic acid, a trichloroacetic acid, a trifluoroacetic acid, an asparatic acid, a glutamic acid. Further, the compounds of the present invention can be isolated as a hydrate, ethanol, isopropanol, or other solvate or various crystalline substance.
The compound of formula (I) or (Ixe2x80x2) according to the present invention may be synthesized by, for example, the following method. 
wherein, R1, R2, R3, R4, R5 and R6 are as defined above and R7xe2x80x2 indicates R7 as defined above except for a hydrogen atom or a protective group of the carboxylic acid such as a benzyl group, an allyl group, etc.
To carry out the present method, a compound (V) is obtained from the compound (VI) according to a known method (for example, J. Med. Chem. 1986, 29, 2363, ibid. 1985, 28, 1558). This reaction causes 1 to 3 equivalents, preferably 1.5 equivalents, based upon the compound (VI), of a trialkylformate such as triethylformate etc. to act on the compound (VI) in 10 to equivalents of acetic anhydride at 100 to 140xc2x0 C. and distills off the solvent after the end of the reaction so as to obtain the desired compound (V). If necessary, the resultant product may be purified by vacuum distillation, etc.
Note that the starting material, that is, the compound (VI), is either a known compound or is obtained from ethyl malonate magnesium salt, 2-chldronicotinic acid, 2,6-dichloronicotinic acid, 2-chloro-6-methylnicotinic acid, or the like in accordance with a known method (for example, J. Med. Chem. 1986, 29, 2363). It is possible to obtain the compound (IV) from the compound (V) obtained according to a known method (for example, J. Med. Chem. 1986, 29, 2363, ibid. 1985, 28, 1558). One equivalent, with respect to the compound (V), of a commercially available (or known) primary alkylamino or heteroarylamine (VII) (for example, aniline, aminonaphthalene, aminopyridine, aminochloropyridine, aminofluoropyridinei nitroaniline, phenylenediamine, etc.) is used in halogenated hydrocarbon such as methylene chloride or aromatic hydrocarbon such as toluene, benzene, or ether such as diethyl ether, tetrahydrofuran, or a mixture thereof at 0xc2x0 C. to room temperature. After the end of the reaction, the resultant product is diluted with an organic solvent, which is not miscible with water, then is successively washed with water and saturated saline. The solvent is then distilled off, whereupon it is possible to obtain the desired compound (IV). If necessary, the resultant product may be purified by column chromatography etc.
The compound (IV) obtained may be processed by a known method (for example, J. Med. Chem. 1986, 29, 2363, ibid. 1985, 28, 1558) to obtain a compound (III). 1 to 1.2 equivalents, based on the compound (IV), of an alkali metal hydride such as sodium hydride, potassium hydride, or lithium diisopropylamide, lithium hexamethyldisilazane, or other strong base, preferably sodium hydride, is used in a halogenated hydrocarbon such as methylene chloride, an aromatic hydrocarbon such as toluene, benzene or an ether such as diethyl ether, tetrahydrofuran, or a mixture thereof at 0xc2x0 C. to room temperature. After the end of the reaction, the resultant product is diluted with an organic solvent, which is not miscible with water, then is successively washed with water and saturated saline. The solvent is then distilled off, whereupon it is possible to obtain the desired compound (III). If necessary, the resultant product may be purified by column chromatography etc.
The compound (III) thus obtained is hydrolyzed according to a known method to obtain a compound (II). The method differs depending on the R7xe2x80x2, but normally can be performed under basic conditions (for example, J. Med. Chem. 1984, 27, 292) or acidic conditions (J. Med. Chem. 1986, 29, 2363).
Under basic conditions, 1 to 1.2 equivalents, based upon-the compound (III), of alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, is used in water or an alcohol such as ethanol, methanol, or ether such as diethyl ether, tetrahydrofuran, dioxane, or a mixture thereof at room temperature to 60xc2x0 C. After the end of the reaction, the reaction solution is made weakly acidic, is diluted with an organic solvent, which is not miscible with water, and is successively washed with water and saturated saline. The solvent is then distilled off to obtain the desired compound (II). If necessary, the resultant product may be purified by recrystallization etc. Under acidic conditions, an acid catalyst such as sulfuric acid, hydrogen chloride, is made to act in water or an alcohol such as ethanol, methanol, or an ether such as diethyl ether, tetrahydrofuran, dioxane, or a mixture thereof at 60xc2x0 C. to 100xc2x0 C. After the end of the reaction, the solvent is distilled off to obtain the desired compound (II). If necessary, the resultant product may be purified by recrystallization etc.
When employing a special substituent group as a protective group of the carboxylic acid in R7xe2x80x2 of formula (VI), it is also possible to convert the substance to the compound (II) by that substituent group under neutral conditions. For example, when employing a benzyl group in R7xe2x80x2, it is possible to convert the substance to the compound (II) by hydrolysis under neutral conditions. When an allyl group is employed in R7xe2x80x2, it is possible to convert it to the compound (II) by formic acid in the presence of a Pd(O) complex. The compound (II) obtained can be used to obtain the compound (Ia) in the compounds having the formula (I) of the present invention where X is the group NR5R6 according to a known method (Fourth Experimental Chemical Seminar, vol. 22, p. 137, published by MARUZEN).
The reaction synthesizes an acid amide from a carboxylic acid (II) and commercially available or known amine component (VIII) (for example, methylamine, ethylamine, isopropylamine, benzylamine, phenylethylamine, aniline, toluidine, aminobenzoic acid, aminoacetophenone, dichloroaniline, aminonaphthalene, aminopyridine, aminodichloropyridine, aminofluoropyridine, phenylenediamine, diaminopyridine, nitroaniline, etc.). This may be done by various methods, but these may be roughly divided into three groups. The first are methods where a condensation agent such as dicyclohexyl carbodiimide, carbonyl diimidazole, is used to cause a reaction betweena carboxylic acid (II) and amine component (VIII). The second are methods where a carboxylic acid (II) is converted to an acid halide, then allowed to react with an amine component (VIII). The third are methods where carboxylic acid (II) is converted to an acid anhydride, then allowed to react with an amine component (VIII).
For example, as a method going through an acid halide, 1 to 5 equivalents, based upon the carboxylic acid (II), of an acid halogenating agent such as thionyl chloride, oxalyl chloride, phosphorus pentachloride, is used a halogenated hydrocarbon such as methylene chloride, chloroform aromatic hydrocarbon such as toluene, benzene, an ether such as tetrahydrofuran, 1,4-dioxane, or a mixture thereof or without using a solvent at room temperature to 100xc2x0 C. After the end of the reaction, the solvent is distilled off to obtain the acid halide. The acid halide may be used as it is, without purifying for the next reaction. 2 to 3 equivalents, based on the acid halide, of the amine component (VIII) is reacted in a halogenated hydrocarbon such as methylene chloride, aromatic hydrocarbon such as toluene, benzene, an ether such as diethyl ether, or a mixture thereof at 0xc2x0 C. to room temperature, or 1 to 1.5 equivalents of the amine component (VIII) is reacted in the presence of 1 to 3 equivalents of an amine such as triethylamine, diisopropylethylamine, pyridine, or the amine component (VIII) may be reacted with the acid halide component after reacting with an alkali metal hydride such as sodium hydride or potassium hydride to form the corresponding amine metal salt. After the end of the reaction, the substance is diluted with an organic solvent which is not miscible with water, then is successively washed with water and saturated saline. The solvent is then distilled off to obtain the desired compound (Ia). If necessary, the product may be purified by column chromatography, recrystallization, etc.
In the process of the present invention, it was possible to introduce R substituent group, which was difficult to be introduced, by the use thereof in the form of the amine.component (VII).
The type IV phosphodiesterase inhibiting activities of the compounds according to the present invention were confirmed by the following test:
(1) Method of Measurement of Type IV Phosphodiesterase Inhibiting Activity
The following assay was used to evaluate the ability of the compound of the present invention to suppress type IV phosphodiesterase, according to Biochemical. Pharmacol. 48 (6), 1219-1223 (1994).
1) Type IV phosphodiesterase activity fractions were prepared as follows. Human histiocytic lymphoma cell line U937 was cultured in an RPMI1640 medium containing 10% fetal calf serum to obtain 109 cells of U937. The cells were recovered by centrifugation and suspended in 40 ml of buffer A (20 mM bis-tris, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, 0.1 mM 4-(2-aminoethyl)benzensulfonyl hydrochloride, 50 mM sodium acetate, pH=6.5). The cells were broken by a sonication and centrifuged (4xc2x0 C., 10,000G, 10 minutes) to obtain a supernatent. This was filtered by a 0.45 xcexcm filter to obtain the soluble fraction.
The soluble fraction obtained was applied into a 1xc3x9710 cm DEAE Sepharose column equalibrated with the buffer A. 120 ml of the buffer A containing a linear gradient solution of 0.05 to 1 M sodium acetate was used to separate the phosphodiesterase and recover 24 5-ml fractions. Each of the fractions was measured for the cAMP phosphodiesterase activity. The fractions having cAMP phosphodiesterase activity which could be inhibited by 30 xcexcM rolipram (selective type IV phosphodiesterase inhibitor) were collected and used as a stored solution for examination of the type IV phosphodiesterase inhibiting activity.
2) The test compound was reacted at a desired concentration in a reaction mixture containing 20 mM tris-HCl (pH 7.5), 1 mM MgCl2, 100 xcexcM EDTA, 330 xcexcg/ml calf serum albumin, 10 xcexcg/ml 5xe2x80x2-nucleotidase, 0.4 xcexcCi 3H-cAMP (0.28 mM cAMP) and the type IV phosphodiesterase stored solution at 30xc2x0 C. for 30 minutes. QAE-Sephadex suspended in 10 mM of hepes-Na (pH-7.0) was added to the reaction mixture which was then allowed to stand for 5 minutes, then the supernatant was obtained, QAE-Sephadex was further added, to the supernatant, and was allowed to stand for 5 minutes, then the supernatant was obtained and measured for radioactivity.
The IC50 was measured for each compound as the concentration of the test compound inhibiting 50% of the type IV phosphodiesterase activity.
(2) Type IV Phosphodiesterase Inhibiting Activity of Various Compounds
The phosphodiesterase inhibiting activity IC50 obtained by the above method of measurement is shown in the following Table I. As the typical control agent, Rolipram (Tocris) was used.
Further, as a Comparative Example, the compound N-(2-(4-pyridyl)ethyl)-1-ethyl-7-methyl-1,4-dihydro[1,8]naphthylidin-4-one-3-carboxyamide described in WO-A-97-04775 (1997), page 17, Example 1 was synthesized and measured similarly for inhibiting activity. The phosphodiesterase inhibiting activity IC50 obtained is shown in Table I.
As a result of the test on the phosphodiesterase inhibiting activity, it was confirmed that the 1-aryl-1,8-naphthylidin-4-one derivative according to the present invention exhibited an excellent inhibitory effect.
The inhibitory activities of the compound of the present invention on TNF-xcex1 production by LPS stimulated macrophages were confirmed by the following test:
(1) Method of Measurement of TNF-xcex1 Production Inhibitory Activity by LPS Stimulated Macrophages
The following assay was used to evaluate the ability of the compound of the present invention to suppress TNF-xcex1 production by LPS stimulated macrophages according to Immuno pharmacol. 29, 121-127 (1995).
1) 6 to 10 week old female BALB/c mice were used, 2 ml portions of thioglycolate were intraperitoneally administered, and the abdominal cavities were washed by 10 ml of PBS after 4 days, whereby 1 to 2xc3x97107 peritoneal cells were obtained per mouse. These were suspended in a hemocyte solution (0.75% ammonium chloride, 17 mM tris-hydrochlorate buffer, pH7.2), centrifuged, then resuspended in an RPMI1640 medium including 10% fetal calf serum and seeded in a 96-well cell culture plate at a density of 1xc3x97105 cells/50 xcexcl/well. Since these cells adhered strongly to the tissue culture plate and were positive in nonspecific esterase staining, they were used for the test as mouse peritoneal macrophages. Mouse peritoneal macrophages were precultured overnight at 37xc2x0 C. in 5% CO2 for the experiment.
2) E. Coli (serum type 055:B5) derived LPS was dissolved in PBS in a concentration of 1 mg/ml, then sterilized by filtration. The test compound was dissolved in DMSO to make a 1000-fold concentration solution of the final concentration of use. 10 xcexcl of the above LPS stock solution (final concentration 10 xcexcg/ml) and 1 xcexcl of the tested substance stock solution were added and mixed in 0.5 ml of RPMI1640 medium containing 10% fetal calf serum. This was added to the above cells at 50 xcexcl/well and cultured for 8 hours. The cultured supernatant was recovered from each well and the TNF-xcex1 concentration was measured by the ELISA method (Cytoscreen(copyright) Immunoassay Kit Mouse TNF-xcex1, BioSource International).
3) The IC50 was calculated for each compound as the concentration of the test compound inhibiting 50% of the TNF-xcex1 production caused by LPS stimulus.
(2) TNF-xcex1 Production Inhibitory Activity by LPS Stimulated Macrophaaes
The IC50 values for the TNF-xcex1 production inhibitory activity obtained by the above method are shown in the following Table II. The comparative example was the compound described in WO-A-97-04775, Example 1, mentioned above.
From the above results, it was confirmed that the compound of the present invention exhibits an excellent activity inhibiting the production of TNF-xcex1.
The compound of the present invention is useful-as a pharmaceutical composition for the prevention or treatment of bronchial asthma, chronic bronchitis, and other respiratory diseases, diseases relating to abnormality of the nervous system such as Alzheimer""s Disease, Parkinson""s Disease, diseases relating to mental abnormalities such as maniac depression, inflammatory diseases such as atopic dermitis, acquired immunity disorder syndrome general or local joint diseases such as osteoarthritis, rheumatoid arthritis, Crohn disease, sepsis, endotoxin shock and other diseases related to tumor necrosis factor (TNF-xcex1) or other various cytokine (IL-1, IL-6, etc.), and the like by selectively inhibiting the type IV phosphodiesterase and further inhibiting the production of TNF-xcex1.
The type IV phosphodiesterase inhibitor of the present invention is useful as an agent for the prevention or treatment of specifically respiratory diseases (for example, bronchial asthma, chronic bronchitis, pneumonia type diseases, adult respiratory distress syndrome, etc.), diseases relating to abnormality of the nervous system (for example, impaired learning, memory, and recognition relating to Alzheimer""s Disease, Parkinson""s Disease, and the like, multiple lateral sclerosis, senile dementia, amyotrophic lateral sclerosis, muscular distrophy, etc.), diseases relating to mental abnormalities (for example, maniac depression, schizophrenia, neurosis, etc.), inflammatory diseases (for example, atopic dermitis, conjunctivitis, acquired immunity disorder syndrome, keloids, etc.), general and local joint diseases (for example, osteoarthritis, rheumatoid arthritis, and other general or local joint diseases, gouty arthritis, rheumatoid arthritis, nodose rheumatism, etc.), tumor necrosis factor (TNF) and other cytokine (IL-1, IL-6, etc.) related diseases (for example, psoriasis, rheumatoid arthritis, Crohn disease, septicemia, sepsis, endotoxic shock, nephritis, pneumonia, bacterial or viral infection, cardiac incompetence, ateriosclerosis, cardiac infarction, etc.) etc.
For use of the effective ingredient of the present invention as a pharmaceutical or a type IV phosphodiesterase inhibitor, one or more types of the compound of the present invention may be formulated and formed into preparations suitable for the method of administration according to ordinary methods. For example, for oral administration, capsules, tablets, granules, powders, syrups, dry syrups, and other preparations may be mentioned, while for nonoral administration, injections and also rectal suppositories, vaginal suppositories, and other suppositories, sprays and other nasal agents, ointments, transdermal absorption type tapes, and other transdermal absorption agents may be mentioned.
The clinical dosage of the compound of the present invention differs depending on the symptoms, the severity of the disease, the age, and complications of the patient to which the compound is being administered and differs depending on the preparation as well, but in the case of oral administration is normally 1 to 1000 mg, preferably 1 to 500 mg, more preferably 5 to 100 mg. per adult per day as effective ingredient, and in the case of nonoral administration is one-tenth to one-half of the case of oral administration. The dosage may be suitably adjusted according to the age, symptoms, etc. of the patient.
The compound of the present invention is a selective inhibitor for type IV phosphodiesterase and has over 10-times the selectivity over other phosphodiesterase isoenzymes (i.e., PDE I-III, V and VII). Due to this, it is expected that there will be few side effects due to the action of inhibiting other phosphodiesterase isoenzymes. The compound of the present invention is low in toxicity. The compound is expected to be high in safety. For example, the compounds of Examples 99, 102, 103, 106, 139 and 141 exhibited no death when 10 mg/kg per day was administered for 28 days to mice.
The 1-aryl-1,8-naphthylidin-4-one -derivative or its pharmaceutically acceptable salt or solvate of the present invention is useful as a pharmaceutical composition for the prevention or treatment of diseases involving type IV phosphodiesterase. As specific examples of diseases involving type IV phosphodiesterase, for example, respiratory diseases (for example, bronchial asthma, chronic bronchitis, pneumonia type diseases, adult respiratory distress, etc.), diseases relating to abnormality of the nervous system (for example, impaired learning, memory, and recognition relating to Alzheimer""s Disease, Parkinson""s Disease, and the like, multiple lateral sclerosis, senile dementia, amyotrophic lateral sclerosis, muscular distrophy, etc.), diseases relating to mental abnormalities (for example, maniac depression, schizophrenia, neurosis, etc.), inflammatory diseases (for example, atopic dermitis, conjunctivitis, acquired immunity disorder syndrome, keloids, etc.), general and local joint diseases (for example, osteoarthritis, rheumatoid arthritis, and other general or local joint diseases, gouty arthritis, rheumatoid arthritis, nodose rheumatism, etc.), tumor necrosis factor (TNF) and other cytokine (IL-1, IL-6, etc.) related diseases (for example, psoriasis, rheumatoid arthritis, Crohn disease, septicemia, sepsis, endotoxic shock, nephritis, pneumonia, bacterial or viral infection, cardiac incompetence, ateriosclerosis, cardiac infarction, etc.) etc. may be mentioned.